The incidence of chronic metabolic diseases such as obesity and diabetes has increased dramatically and constitutes one of the major threats to global health. Recent studies have indicated that cell stress response plays an essential role in the pathogenesis of type 2 diabetes (T2D). Oxidative stress and endoplasmic reticulum (ER) stress cooperatively promote cell dysfunction, apoptosis and insulin resistance. While the activators and downstream effects of cell stress have been partially characterized, much is unknown regarding the molecular mechanisms by which the cell stress responses are regulated. Our lab and others have recently characterized the NLR (NBD-LRR) family of proteins, which have been shown to mediate the cell stress response to microbes and environmental stressors. My preliminary data indicates that NLRX1, a mitochondria-localized NLR protein, promotes ER stress response by mediating the generation of mitochondrial reactive oxygen species (mROS). Mechanistically, NLRX1 directly associates with ECSIT (evolutionarily conserved signaling intermediate in Toll pathways) and TRAF6 (TNF receptor-associated factor 6), both of which are important in mitochondrial respiratory chain assembly. NLRX1-deficient (Nlrx1-/-) cells are protected from ER stress-inhibited insulin-PI3K-Akt pathway. Moreover, Nlrx1-/- mice were protected from obesity- induced insulin resistance by high-fat diet (HFD) feeding. Therefore, I hypothesize that NLRX1 mediates mROS generation by facilitating ECSIT-TRAF6 function, which subsequently promotes ER stress, and that the NLRX1-mediated cell stress responses impair insulin signaling in insulin target tissues. I will employ T2D (HFD feeding and leptin-deficient ob/ob mice) animal models to examine the function of NLRX1-mediated cell stress responses in insulin resistance. I will examine how NLRX1 controls ECSIT function and mROS generation. The goal of the proposal is to examine the mechanism of the interaction between NLRX1-mediated oxidative stress and ER stress in promoting cell dysfunction and a defect in insulin signaling. The proposed genetic and biochemical analyses and animal model studies will provide novel insights into the regulation and function of metabolic signaling pathways. Further studies could lead to the identification of new therapeutic targets and ultimately help develop rational, mechanism-based treatment strategies that target obesity and diabetes.